Bottom Line:
MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes.To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy.As proof of principle we have used mir-130a and its validated target MAFB to test this strategy.

ABSTRACTMicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.

gks145-F2: MCF7 cells were transfected with the plasmid library and selected in 500 μg/ml zeocin. Zeocin resistant and normal MCF7 cells were selected in different concentrations of GCV showing GCV sensitivity of the library transfected cells.

Mentions:
Initially three constructs of a selectable marker fused in-frame to HSVTk were tested for resistance to the selectable marker and sensitivity to GCV. The constructs used were HyTK, TKpuro and TKzeo conferring resistance to hygromycin, puromycin and zeocin respectively. TKzeo conferred the highest sensitivity to GCV and was therefore selected for the library construction (Supplementary Figure S3). To analyse the GCV sensitivity of the zeocin-resistant cells normal MCF7 cells and zeocin-resistant cells were plated and incubated with different concentrations of GCV. After 10 days the plates were stained, clearly showing that untransduced MCF7 cells are resistant to GCV up to the highest concentration, 24 μM, tested (Figure 2). In contrast, zeocin resistant MCF7 cells were sensitive to GCV even at the lowest concentration, 2 μM, tested. Conversely, it was shown that down-regulation of TKzeo by binding of mir-130a to the MAFB UTR cloned downstream of TKzeo resulted in zeocin sensitivity of these MCF7 cells (Supplementary Figure S4). To test the stability of the sensitivity to GCV over time of the zeocin-resistant MCF7 cells, zeocin was removed at Day 0 and at Days 1, 30 and 60 after zeocin removal cells were selected in 8 μM GCV for 10 days. The results show that in cells that had been without zeocin selection pressure for 30 or 60 days false positive GCV-resistant clones appear. However, in cells that have only been without zeocin for 1 day no GCV-resistant colonies develop. Renewed zeocin selection at Day 60 for 10 days followed by GCV selection restored GCV sensitivity to the cells (Supplementary Figure S5).Figure 2.

gks145-F2: MCF7 cells were transfected with the plasmid library and selected in 500 μg/ml zeocin. Zeocin resistant and normal MCF7 cells were selected in different concentrations of GCV showing GCV sensitivity of the library transfected cells.

Mentions:
Initially three constructs of a selectable marker fused in-frame to HSVTk were tested for resistance to the selectable marker and sensitivity to GCV. The constructs used were HyTK, TKpuro and TKzeo conferring resistance to hygromycin, puromycin and zeocin respectively. TKzeo conferred the highest sensitivity to GCV and was therefore selected for the library construction (Supplementary Figure S3). To analyse the GCV sensitivity of the zeocin-resistant cells normal MCF7 cells and zeocin-resistant cells were plated and incubated with different concentrations of GCV. After 10 days the plates were stained, clearly showing that untransduced MCF7 cells are resistant to GCV up to the highest concentration, 24 μM, tested (Figure 2). In contrast, zeocin resistant MCF7 cells were sensitive to GCV even at the lowest concentration, 2 μM, tested. Conversely, it was shown that down-regulation of TKzeo by binding of mir-130a to the MAFB UTR cloned downstream of TKzeo resulted in zeocin sensitivity of these MCF7 cells (Supplementary Figure S4). To test the stability of the sensitivity to GCV over time of the zeocin-resistant MCF7 cells, zeocin was removed at Day 0 and at Days 1, 30 and 60 after zeocin removal cells were selected in 8 μM GCV for 10 days. The results show that in cells that had been without zeocin selection pressure for 30 or 60 days false positive GCV-resistant clones appear. However, in cells that have only been without zeocin for 1 day no GCV-resistant colonies develop. Renewed zeocin selection at Day 60 for 10 days followed by GCV selection restored GCV sensitivity to the cells (Supplementary Figure S5).Figure 2.

Bottom Line:
MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes.To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy.As proof of principle we have used mir-130a and its validated target MAFB to test this strategy.

ABSTRACTMicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.